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Targeting an hiv-1 nef-host cell kinase complex

Active Publication Date: 2011-08-04
UNIVERSITY OF PITTSBURGH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]By the present invention, are provided compounds that inhibit the kinase activity of a Nef:SFK complex, for example, without limitation the Nef:Hck complex, which compounds were identified by screening assays described herein. The terms “derivative thereof,”“mutant thereof” and like terms in the context of proteins contemplated by the present invention (e.g., Nef, Hck, Hck-YEEI, and the like) refers to mutation (e.g., deletion, addition, and/or replacement of amino acids) which mutation does not eliminate the utility of the resulting proteins in the assays described and provided herein. Thus, the “mutation” contemplated by the present invention includes conserved amino acid replacement with respect to charge type (e.g., Glu/Asp or Lys/Arg interchange and the like), lipophilicity (e.g., Leu/Ile interchange and the like), side chain bulk (e.g., Phe/Tyr interchange and the like), and other metrics that are well known to protein chemistry. “Mutation” also contemplates deletion and insertion of amino acid residu

Problems solved by technology

To reiterate, lack of a catalytic function makes analyses of the interaction of Nef with small molecule inhibitors by a variety of approaches, such as high-throughput screening (HTS) approaches, problematic.

Method used

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  • Targeting an hiv-1 nef-host cell kinase complex
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Examples

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Effect test

example 1

Recombinant Protein Expression and Purification

[0127]Hck-YEEI and Nef were expressed in Sf9 insect cells and purified as described, for example, by Trible, 2006, Id.

example 2

In Vitro Kinase Assay and Chemical Library Screening

[0128]Protein-tyrosine kinase assays were performed in 384-well plates using the Z′-LYTE™ kinase assay system and “Tyr 2 peptide” substrate (Invitrogen, Carlsbad, Calif.) as described by Trible et al. (Id.) Chemical libraries were purchased from ChemDiv, Inc. (San Diego, Calif.) and include a kinase-directed library (2500 compounds), a phosphatase directed library (2500 compounds) and a diversity set (5040 compounds). Library screens were conducted 384-well plates in a final volume of 10 μl per well. Compounds were added to each well (10 μM final), followed by a preformed complex of Hck-YEEI (10 ng / well) and Nef (1:20 molar ratio) plus the substrate peptide (2 μM). Reactions were initiated by the addition of ATP (50 μM final) and incubated at room temperature for 35 minutes. Reactions were developed and terminated per manufacturer's protocol, and with fluorescence ratios calculated as described (Trible et al., Id.)

example 3

HIV Replication Assay

[0129]HIV-1 replication assays were conducted using HIV-1 strain NL4-3, a strain that is very similar, as known to those of skill in the art, in sequence to the SF2 allele used in the yeast assays and that strongly activates Hck-YEEI. Virus stocks were prepared by transfection of the recombinant viral genome into 293T cells. Viral replication was monitored in the U87MG astroglioma cell line expressing CD4 and CXCR4 (Salvatori, F. & Scarlatti, G., 2001, AIDS Res. Hum. Retroviruses 17: 925-35; Trkola, A. et al., 1998, J. Virol. 782:1876-1885). Viral replication was monitored by measuring p24 protein levels in the culture supernatant 4 days after infection by standard ELISA-based techniques. Test compounds were added to the culture 30 min prior to infection with HIV, and DMSO was used as the carrier solvent at a final concentration of 0.1%.

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Abstract

Drug candidates for inhibition of HIV-I replication can target Src family kinases (SFK), such as Hck, that interact with Nef protein of the virus. Compounds characterized by such inhibitory activity were identified via an assay for kinase activity of an SFK in a Nef:SFK complex. Illustrative of inhibitors identified using the kinase assay are various 2,3-diaminoquinaxolines and furo[2,3-d]pyrimidines. The inventive inhibitors were found to arrest HIV-I viral replication in vitro.

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS[0001]This application claims priority from U.S. Provisional Application Ser. No. 61 / 053,135, filed May 14, 2008, incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH[0002]This invention was funded by NIH AI57083 and NIH CA81398 grants. The United States government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]The present invention relates generally to inhibition of protein activity, and in particular kinase activity, and compounds and assays for measuring such inhibition useful for identifying novel inhibitors of kinas activity. In particular, the present invention relates Nef protein and inhibition of its interaction and complexes comprising Nef, with other cellular components as described herein and known in the art.[0004]The information provided herein is intended solely to assist the understanding of the reader. None of the information provided nor references cited ...

Claims

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Application Information

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IPC IPC(8): A61K31/635C07D241/44C07D405/12C07D491/048A61K31/498A61K31/519A61K31/5377A61P31/18
CPCC07D241/44C07D491/048C07D491/04C07D405/12A61P31/18
Inventor EMERT-SEDLAK, LORIKODAMA, TOSHIAKIDAY, BILLY W.DAI, WEIXIANGTRIBLE, RONALD P.SMITHGALL, THOMAS E.
Owner UNIVERSITY OF PITTSBURGH
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